Compounds of 2&#39;,4&#39;-substituted anilides of substituted nitrosalicylic acid

ABSTRACT

1. A 3-TERT. BUTYL-6-METHYL-5-NITROSALICYLANILIDE OF THE FORMULA:   1-(CH3-C(-CH3)2-),2-(HO-),3-(R2-(3-R1-1,4-PHENYLENE)-   NH-CO-),4-(CH3-),5-(O2N-)BENZENE   IN WHICH R1 IS H, HALO, CF3 OR METHYL AND R2 IS CHLORO, BROMO, IODO OR CF3.

United States Patent 3,839,443 COMPOUNDS OF 2',4'-SUBSTITUTED ANILIDESOF SUBSTITUTED NITROSALICYLIC ACID William H. Meek, Northfield, Ohio,assignor to Ferro Corporation, Cleveland, Ohio No Drawing.Continuation-impart of application Ser. No. 186,514, Oct. 4, 1971. Thisapplication Aug. 1, 1972, Ser. No. 277,076

Int. Cl. C07c 103/26 US. Cl. 260-559 S Claims ABSTRACT OF THE DISCLOSUREA class of organic compounds is disclosed comprising substitutedanilides of 3-tert. butyl-6-methyl-5-nitrosalicylic acid, characterizedin that the anilide portion is substituted only in the 4 position oronly in the 2, 4' positions. Moreover, the substituents themselves forthe anilide portion must be selected from a relatively small number ofmonovalent substituents. Such compounds are useful in the control ofmicroorganisms and especially as anthelmintics, that is, therapeuticagents for destroying parasitic life, such as intestinal Worms. When thesubstituents for the 4' position or the 2, 4' positions are furtherrestricted to a still smaller, select number, the resulting compoundshave markedly superior results as anthelmintics as compared to othercompounds of the present invention.

CROSS-REFERENCE TO RELATED APPLICATION This application is acontinuation-in-part of an application, entitled 3-Tert.Butyl-6-Methyl-5-Nitrosalicylic Acid, and Esters, and Amides Thereof,filed Oct. 4, 1971, and assigned Ser. No. 186,514. The parentapplication discloses and claims, among other compounds, substitutedanilides of the defined nitrosalicylic acid.

BACKGROUND OF THE INVENTION The need for combating the growth ofundesirable microorganisms, bacteria, insects, and the like is acontinuing and increasing one. Many organic compounds have beensuggested as a detergent to such growth or as an effective destroyer ofthe undesired life.

As compared to a mere killing or destroying of microorganisms, etc., aquite different situation prevails when one is concerned with killingonly one of two cohabiting classes of living things without harming theother. A common example of this is undesirable parasitic infestation ona desirable, living animal.

More specifically, various types of worm parasites are found in mammalsof commercial importance to man. The most important are the parsites oflivestock, especially of ruminants, such as sheep, goats, and cattle.However, other ruminants are similarly affected such as oxen, deer,water buffalo, etc. The more significant parasites are the nematodes ofthe alimentary tracts and the trematodes (flukes) which infect theliver. The alimentary tract nematodes are principally important insofaras they reduce the growth of the host animals and render less efficientthe consumption of feed by the animals. The trematodes directly affect avital organ and can cause severe illness and death in the host animal.

Obviously, a treating agent which not only kills the parasites, but alsokills the host animal is of no utility. Conversely, a treating agentthat is harmless on the host animal but only slightly retards the growthof parasitic life is of little real value. What is needed is a treatingagent that not only efficiently kills or expels the parasites, but whichis harmless to the host; or for which the host has a large margin oftolerance, that is, an agent of which the host can take massive dosageswith little or no harm.

Patented Oct. 1 1974 Moreover, although both types of mentionedinfestations, the nematodes and the trematodes, occur commonly andnaturally in the same types of livestock, presently known medicationsnormally used for control on one of these infections is generallyineffective for the control of the other.

The matter of tolerance of a host animal for a therapeutic agent, suchas an anthelmintic, cannot be overemphasized, especially when the animalmust be administered to from a group or herd of animals in anunavoidably somewhat imprecise manner. In the treatment of large groupsof animals, there is serious risk that some animals may be inadvertentlytreated more than once, and thus subjected to double or triple dosage;that some animals will be overdosed because of errors in estimatingtheir individual weight; that some animals will, by virtue of individualgenetic variation, and variable state of health or debility, have lesstolerance than the average animal for any medication. It is thereforeclearly desirable that the typical animal be able to tolerate Withoutserious harm, as large a multiple as possible of the minimum dosageregarded as likely to be effective as an anthelmintic.

The difference in activity between an effective amount of ananthelmintic on parasites and on a host animal can be quantitativelyexpressed as a Therapeutic Index. This index is defined as the maximumdose at which no toxic symptoms in the host animal are observed, dividedby the minimum dose at which the anthelmintic is therapeuticallyeffective. In general, an anthelmintic is considered to betherapeutically effective against a given parasite when it kills orexpells from the host at least and preferably close to of the viableforms of that parasite.

SUMMARY OF THE INVENTION It has now been discovered that a class ofcompounds comprising substituted anilides of 3-tert.-butyl-6-methyl-S-nitrosalicylic acid have particular utility as a control for varioustypes of microorganisms and also make effective anthelmintics againstboth nematodes and trematodes when substituted only in the 4 position onthe anilide moiety, or when substituted only in the 2 and 4' positions,and no other positions. Additionally, the substituents themselves mustbe selected from a rather limited class. Still further, when thesesubstituents are reduced to an even more limited class, the resultingcompounds make superior anthelmintics. The latter compounds are not onlyhighly efficient in killing a wide spectrum of parasites but can betolerated without harm by host animals in relatively large amounts.

A compound of the present invention has the general formula:

in which R is H, halo, CF or alkyl of 1 to 4 carbon atoms, and R ishalo, CF or CN.

Compounds of the indicated formula exhibit marked improvement foranthelmintic activity when R is limited to C1 or CH and R is limited toCl, as compared to the other defined compounds of Formula I.

3 DESCRIPTION OF THE PREFERRED EMBODIMENTS Here and in the claims thefollowing number system of salicylanilides is used:

The salicylic acid portion of all compounds of the present inventionhave the same molecular configuration to afford the most usefulcompounds, namely, hydroxyl at the 2 position as indicated, tert. butylat the 3 position, nitro at the 5 position, and methyl at the 6position. The anilide portion of all compounds of the present inventionare substituted in only one or only two specific positions and at noother positions, namely, at the 4 position only or at the 2' and 4'positions only.

Additionally, the substituents at the 2, 4 positions must be selectedfrom a relatively small group of monovalent substituents. As indicated,referring to the Formula I of the preceding section, R must be H, halo,CF or alkyl of l to 4 carbon atoms, and R must be halo, CF or CN.Anthelmintics of superior properties and performance are obtained when Ris restricted to C1 or CH and R is restricted to Cl.

Preparation of compounds of the present invention are described in thecited parent application, Ser. No. 186,514. In general,3-tert.butyl-6-methylsalicylic acid may be nitrated in toluene solutionwith dropwise addition of nitric acid for about an hour at about 65 C.to about 70 C. Upon cooling, 3-tert. butyl-6-methyl-5-nitrosalicylicacid precipitates and may be recovered by filtration.

The anilide of the described salicylic acid may then be formed byreacting the acid with a phenylamine, the phenyl nucleus having thedesired substituents or substituents. This reaction may be carried outin toluene solution at 110 C. under reflux conditions for four to sixhours in the presence of a condensing agent such as POCl or PCl Uponcooling, a precipitate of a substituted anilide of 3-tert.butyl-6-methyl-5-nitrosalicylic acid forms which can be recovered byfiltration, washing, and drying.

The new method of controlling helminths or parasitic worms by thisinvention comprises administration to a host animal such as a ruminantanimal in need of such treatment orally alone, combined with apharmaceutical or a feed carrier, an effective anthelmintic butnon-toxic quantity of a compound of Formula I. In certain cases such asextra gastrointestinal infestation of flukes in sheep, the compounds maybe administered parenterally, i.e., in a sterile micronized suspensionor solution.

The administration is in quantities nontoxic but effective either forcurative or prophylactic purposes and has broad range of activity ongastrointestinal parasites of warm blooded animals especially sheep andcattle. The hclminths most effectively treated with the new compoundsare the Trematodes, Cestodes or, and especially, Nematodes. Activityagainst flukes such as Fasciola or Fasciola hepatica is alsoparticularly pronounced as noted above. More specific parasiticinfestations in which this invention may be applied are found in theMerck Veterinary Manual, Third Edition, pages 699-806, as are generalmethods of control of internal parasites; see also U.K. 1,183,641.Generally effective closes range from about 1 up to about 50 mg./kg. ofbody weight, preferably about 5 to mg./kg. Effective doses in sheepwithout significant side effects have been found to be about 1 to 50mg./kg. Most usually the dosage unit compositions are administered from1 to 5 times daily, preferably for convenience one treatment is used toclear the infection.

Veterinary or composition containing sufficient quantities of thecompounds of Formula I to reach the dose levels mentioned above areprepared as known to the art by preparing tablets, capsules, boluses,liquid suspensions,

powders, drenches or solutions for injection in packaged form.Alternatively, especially for prophylaxis, premix or feed compositionscontaining effective but nontoxic quantities of the activesalicylanilide are used. For these purposes particulate carriers, inertpowders or, especially, feed carriers such as soybean meal, corn oil,vermiculite, diatomaceous earth, barley or wheat are used. In dosageunit or premix feed compositions the compound can comprise from about 5to 75% of the final composition as is convenient for the farmer orveterinarian. As an example, a 5% salicylanilide-vermiculite or soybeanmeal premix can be used which will be uniformly mixed with the animalfeedstuff. Alternatively, a lick or pasture block can be used for fieldanimals.

To show the relative effect of compounds of the present inventiondiffering in the R and R substituents of Formula I, as well as to showthe relative effect of those compounds with respect to still otherrelated compounds which are not of the present invention, that is, inwhich R and R are monovalent substituents other than those claimed, aseries of tests were carried out on living animals as follows.

Two groups of sheep were used in the tests. One group was used as anon-medicated control, while the other group was treated with thecompound under investigation. Helminth-free young lambs were infectedwith about 10,000 filariform larvae of Haemonchus contortus. In threeweeks when the infection became apparent, egg counts were made todetermine the density of the worm burden.

The egg count was carried out using the following modified Stoll method.Three grams of sheep feces were taken rectally from each animal andplaced in a Stoll flask. To each flask, tap water was added to the topline. The fecal pellets were emulsified using a narrow tongue depressor.If the pellets were too hard to break down, the flasks were closed withrubber stoppers and kept in the refrigerator for several hours. Theflasks were thoroughly shaken and a 1 mi. sample was drawn from themiddle using a wide mouth pipette. This 1 mi. sample was transferred toan 8 ml. plastic tube, to which enough saturated sodium chloridesolution was added to reach the rim. The tubes were then centrifuged forten minutes at 1500 rpm. then sodium chloride solution was added with amedicine dropper forming a concave miniscus. A cover glass was carefullyadded and the tubes were kept in a refrigerator for ten to twentyminutes. During that period, the nematode ova flowed upward and adheredto the bottom of the cover glass slip. At the end of the 20-minuteperiod, the cover slip was carefully detached from the miniscus andtransferred to a microscpic slide. The ova were then counted under amicroscope equipped with 10X objective lens. Each ovum under themicroscope represented 20 ova in the sample.

Animals with sufficiently high egg counts were taken out of the pool andhoused in experimental pens. Since the salicyianilides of the presentinvention are water insoluble, the compounds were first ground using amortar and pestle and then were placed in a carboxymethyl cellulosesolution. The particles were further micronized ultrasonically. Thecompound was then administered to two or three animals by Oesophagealgavage. Control, untreated animals, received only the carboxymethylcellulose solution in a similar manner. On the sixth and seventh dayafter treatment, fecal pellets were taken from the rectum of the controlas well as the treated animals and egg counts were made.

Tests for activity against live flukes were conducted as follows:

Eggs of Fasciola Izepatica were collected from the bile of donor sheep.The eggs were embryonated and snails were infected (the genus Lymnaeaserves as intermediate host) to produce Metacercariae which are theinfective forms for sheep. Each sheep was infected with 250metacercariae intraruminally. When the infection became patent in ca. 80days, egg counts were carried out to determine the degree of wormburden. The liver was processed to recover F. hepatica in the untreatedand treated sheep.

The following examples illustrate the present invention and should notbe construed as imposing limitations upon the claims. Percentages are byweight percent unless otherwise indicated.

Although the present compounds are uniquely adapted for use asanthelmintics, they also find use in the control of various types ofmicroorganisms. The following Example 1 illustrates such use to controlmicroorganisms; Example 2 illustrates the use of the present compoundsas a sheep drench; Example 3 describes the preparation of a ruminantbolus; Example 4 shows the elfect of the present compounds on liverflukes; Examples 5 through 13 provide results of the present compoundswhen used as anthelmintics; Examples 14 through 40 provide comparativeresults of related compounds having substituents differing from thoseclaimed; and Examples 40 through 49 provide physical data of some of thepresent compounds.

Example 1 The following procedure for antibacterial evaluation was used.Test compounds were dissolved in a suitable solvent (typically dimethylsulfoxide or acetone) and incorporated in a nutrient agar at variousconcentrations. The plates were then streaked with cultures of theappropriate bacteria. The inoculum contained about 8 l0 organisms permilliliter. After incubation for 48 hours at 37 C., the plates wereexamined for evidence of growth of the microorganisms. The minimumconcentration necessary for complete inhibition of growth was noted.

For testing against the fungus Trichophyton the same procedure was used,except that the test medium was Sabouraud agar, and the plates werecultured for 120 hours at 25 C. before evaluation for evidence ofgrowth. The inoculum added to the culture media contained about 5 sporesin a water suspension containing 0.1% peptone.

Thus 4'-bromo-3-tert. butyl-6-methy1 5 nitrosalicylanilide was observedto have the following minimum inhibitory concentrations in agar platetests against bacteria.

Minimum Inhibitory Concentration (P.p.m.) Staphylococcus aureus 1Haemophilus gallinarum 8 Escherichia coll 64 Streptococcus foecalis 64Salmonella cholerasuis 64 Salmonella gallinarum 64 EXAMPLE 2 SheepDrench Parts by Weight 3-tert. Butyl-2',4'-dichloro, 6-methy15-nitrosalicylanilide Terra alba 75.5 Tragacanth 3.0 Sodium laurylsulfate 1.5 Water The above solid components are mixed to give awaterdispersible powder to be used on concentrations of 5 g. of powderto 5 ml. of water. The drench is used orally as necessary and practicalto control gastrointestinal infections.

EXAMPLE 3 Ruminant Bolus Grams 3-tert. Butyl-4'-chloro-2', G-dimethylS-nitrosalicylanilide 0.5 Calcium phosphate 4.0 Maize starch 0.54 Talcum0.14 Gum arabic 0.15 Magnesium stearate 0.05

The phosphate and salicylanilide are mixed and screened, then granulatedusing one-half the starch. The screened and dried granules are mixedwith the remaining ingredients, blended thoroughly and compressed on abolus press.

Similarly, tablets can be prepared with reduced fillers.

EXAMPLE 4 The compounds of this invention also have utility againstliver flukes. When administered an oral dosage of 5 milligrams perkilogram of body weight of sheep infected with liver flukes of the genusFasciola, '3-tert. butyl- 2.,4 dichloro-6-methyl-5-nitrosalicylanilide,completely destroyed all flukes within three days. Again immature flukeinfestations in sheep similar activity was found at 15 mg./kg. The sameeffects against mature flukes were observed when 3-tert. butyl-4'-chloro-Z', 6-dimethyl-5- nitrosalicylanilide was administered at 5milligrams per kilogram of body weight. The 4' cyano congener was activeagainst mature cflukes at 2.5 and 5 mg./kg.; the 4' bromo was active at15 mg./kg. By contrast the isomeric compound 3-tert. butyl-3'-chloro 2,6-dimethyl-5- nitrosalicylanilide is ineffective against liver flukeseven when the sheep receive 15 milligrams per kilogram of body weight.

Other compounds of this invention which were tested and foundefficacious at 15 milligrams per kilogram of sheep body weight were:3-tert. butyl-6-methyl-5-nitro-4'- trifluoromethylsalicylanilide and3-tert. butyl-4-chloro-6- methyl-5-nitrosalicyianilide.

In respect, compounds of this invention differ from compounds currentlycommercial use which are not effective agaist both gastrointestinalworms and liver flu'kes. Since the simultaneous occurrence of both formsof infections is common in commercial ruminant husbandry, the value of asingle form of medication to cure both types of infection isself-evident.

EXAMPLES 5 THROUGH 13 The results of tests on sheep in a mannerdescribed, supra, using compounds of the present invention are given inTable 1, Examples 5 through 13. The numbers in the column headedHaemonchus (15 mg./kg.) refer to the percent of parasites destroyed orexpelled when the indicated compound was introduced into the rumen ofsheep at a dosage of 15 milligrams per kilogram of body weight. Thevalues under Tolerance (mg/kg) refer to the highest test dosage in themilligrams of anthelmintic per kilogram of body weight at which thesheep developed no toxic symptom.

The broken line extending across the table separates two classes ofanthelmintics of the present invention. The compounds represented abovethe line are not only highly efiicient in killing or expellingHaemonchus, but they are tolerated by sheep in relatively large dosages.The compounds represented below the broken line are less attractiveeither because the tolerance for these compounds is less (Examples 8,ll, 12), because they are somewhat less elfective at normal dosages(Examples 9, 10), or because they are inherently more costly to produceand therefore potentially less effective per unit costs to the livestockproducer (Example 13). Because of this the data on tolerance have beenless thoroughly studied. However such compounds are still useful, and inthe cases of specific variation of livestock or under special condition7 8 of cost and availability, they can be attractive antpoor. Table IIsummarizes the data for such other substithelmintic compounds. uents.

TABLE I substituents Haemonchus Tolerance Example R1 R1 (2 mgJkg.)rug/kg.) mg./kg.) (mg/kg.)

It will be noted that the combination of substituents TABLE II listedfor R and R above the broken line are not only Effectiveness 100%effective in killing or expelling Haemonchus, but in removal ofHaemonchus at dosages greater than 50 milligrams per kilogram of bodyanintmmrgleml weight and less than 100 milligrams per kilogram ofExample R1 R2 dg s agf gls body weight are tolerated oy the sheepwithout harmful H 0 effects. substituents of H and F in the 2' and 4'positions, H 0 respectively, were 100% effective in killing or expellingg Haemonchus and tolerated in dosages in excess of 100 H 0 SO NHz 0milligrams per kilogram of body weight. 002mm 18 In contrast to the goodactivity of the 2', 4'-dichloro 8' s congener (Example 5) 100% at 5mg./-kg. and tolerance at and 100 mg./kg., an isomeric compound of theprior art namely the 2, 5'-dichloro (see Monsanto British Pat. No.1,252,087 published Nov. 3, 1971, first compound on page 5) has only 61%activity at 5 mg./ kg. and shows toxic elfects at 50 mg./ kg. It will beappreciated that only a reduction in worm burden is not a practical orcommercial objective for a new anthelrnintic. The Monsanto patent alsodiscloses the salicylanilides only as larvicidal compounds againstLepidoptera or chewing insects. It discloses no activity againstinternal parasites such as pin worms, round worms, flukes, etc. where aprincipal use of this invention lies.

EXAMPLES 23 THROUGH 26 The importance of the 2, 4' substitution on theanilide moiety of Formula I is illustrated by the case of the dichlorosubstitution as summarized in Table III. It is evident from these datathat the shift of one or both of the chlorine atoms from the 2', 4configuration increases toxicity and/or decreases efficacy. This hasbeen mentioned briefly hereinabove. As used herein, toxic refers todeath of sheep resulting from the use of the indicated compound at theindicated dosage. The 3, 5 dichloro compound was not tested fortolerance since its effectiveness was too low to be useful.

26 3,5-diehloro EXAMPLES 14 THROUGH 22 When compounds of Formula I wereused on sheep having substituents in the 2' and -4' positions, otherthan EXAMPLES 27 THROUGH 30 Similarly, a shift of the substituents forthe 2'-methyl- 4'-chloro-substituted anilide moiety of Formula Imaterially reduces efiicacy and safety as shown by the following thoseherein disclosed and claimed, the results were quite Table IV:

TABLE IV Efl'ectlveness ercent R1 and R, (p Tolerance substituents 5mg./kg. 15 mgJkg; 50 mgJkg. 100 mgJkg.

Nontoxie Toxic.

-3chl0ro.- 87 Toxic 2-methyl-5-chloro. 0 2-methyl-6-chloro 0 Thetolerances of Examples 29 and 30 were not determined, since thecompounds were ineffective in combating the parasites in sheep.

EXAMPLES 31 THROUGH 35 For compounds of the present invention (FormulaI) having hydrogen in the 2' position of the anilide moiety and halogenor trifiuoromethyl in the 4' position, the elfect of moving the4'-substituent is shown by the following Table V:

TABLE V Effectiveness at 15 mgJkg. against Haemonchns 4-substitu-3-substitu- 2-substitu- Example substituent tion tion tion Thus, it isclear that a special and unpreducted superiority is associated withsubstitution in the 4-position.

EXAMPLES 36 THROUGH 40 The addition of a third substituent to a 2',4'-dichloro or 2'-methyl-4-chlorosubstituted anilide moiety is alsodisadvantageous as shown by the following Table VI:

EXAMPLES 41 THROUGH 49 The following Table VII provides physicalcharacteristics including color and melting points of some of thecompounds of the present invention. These compounds are solids and wereprepared by the process described herein. The compounds are identifiedby their *R and R substituents in accordance with Formula I.

TABLE VII Substituents Melting Color point, C.

Pale yellow .-.do

While the foregoing describes several embodiments of the presentinvention, it is understood that the invention may be practiced in stillother forms within the scope of the following claims.

-I claim:

1. A 3-tert.butyl-6-methyl-5-nitrosalicylanilide of the formula:

IN H):

in which R is H, halo, CF or methyl and R is chloro, bromo, iodo or OP}.

2. The compound of claim 1 in which R is other than 3. The compound ofclaim 1 in which R is CE, and R is chloro.

4. The compound of claim 1 in which R and R are chloro.

5. The compound of claim 1 in which R is methyl and R is chloro.

References Cited UNITED STATES PATENTS 3,674,849 7/ 1972 Darlington etal 260-559 3,216,896 11/1965 Early et al 260-559 FOREIGN PATENTS 660781012/1966 Netherlands 260-559 HARRY I, MOATZ, Primary Examiner US. Cl.X.R.

1. A 3-TERT. BUTYL-6-METHYL-5-NITROSALICYLANILIDE OF THE FORMULA: